Dag-type and indirect protein kinase c activators and anticoagulant for the treatment of stroke

ABSTRACT

The present disclosure provides a method for treating stroke by administering an anticoagulant, e.g., recombinant tissue plasminogen activator (rTPA), and a protein kinase C (PKC) activator, wherein the PKC activator may be administered before, after, or at the same time as the rTPA. The methods disclosed herein may limit the size of infarction and/or reduce mortality, the disruption of the blood-brain barrier, and/or the hemorrhagic damage due to ischemic stroke compared with rTPA administration alone; and may also extend the therapeutic time window for administering rTPA after a stroke. Also disclosed are compositions and kits comprising rTPA and a PKC activator for treating stroke.

This application claims priority to U.S. Provisional Application Nos. 61/362,464 filed Jul. 8, 2010, 61/412,753 filed Nov. 11, 2010, and 61/412,747 filed Nov. 11, 2010, the entire disclosures of which are incorporated by reference herein.

The present disclosure relates generally to administration of an anticoagulant, e.g., recombinant tissue plasminogen activator (rTPA), and a protein kinase C (PKC) activator to treat a subject following ischemic stroke. The methods disclosed herein may limit the size of infarction and/or reduce mortality, the distruption of the blood-brain barrier, and/or the hemorrhagic damage due to ischemic stroke compared with rTPA administration alone. The methods disclosed herein may also extend the therapeutic window in which rTPA can be administered following a stroke and still be efficacious. Compositions and kits comprising rTPA and a PKC activator are also disclosed.

Stroke

Stroke, also known as a cerebrovascular accident (CVA), is a medical emergency and can cause permanent neurologic damage or even death if not promptly diagnosed and treated. It is the third leading cause of death and the leading cause of adult disability in the United States and industrialized European nations. On average, a stroke occurs every 45 seconds and someone dies every 3 minutes. Of every 5 deaths from stroke, 2 occur in men and 3 in women.

A stroke is an acute neurological injury in which the blood supply to a part of the brain is interrupted, leading to the sudden loss of neuronal function. The blood supply to the brain may be interrupted in several ways; the disturbance in perfusion is commonly arterial, but may be venous.

Different types of stroke include ischemic stroke and hemorrhagic stroke. Ischemic stroke or cerebral ischemia is caused by a temporary or permanent restriction of cerebral blood flow and oxygen supply caused by, for example, an embolis (embolic stroke) or blood clot (thrombolyic stroke). In contrast, a hemorrhagic stroke is caused by the blood vessel rupture (e.g., ruptured aneurysm), which leads to severe bleeding in the brain.

In stroke, the part of the brain with disturbed perfusion no longer receives adequate oxygen (hypoxia). This initiates an ischemic cascade causing brain cells to die or be seriously damaged, thereby impairing local brain function. A transient ischemic attack (TIA) or “mini-stroke” normally lasts less than 24 hours, but is associated with the same symptoms as stroke such as sudden numbness or weakness of the face, arm, or leg; sudden confusion, trouble speaking or understanding; sudden trouble seeing in one or both eyes; and/or sudden trouble walking, dizziness, loss of balance or coordination. Typically, TIAs do not result in permanent brain injury through acute infarction (i.e., tissue death) but they may indicate serious risk of subsequent stroke. An infarctive stroke typically involves a more severe vessel blockage that can last longer than 24 hours without intervention. Cerebral infarctions vary in severity; about one third of the cases result in death.

Ischemia may be confined to a specific region of the brain (focal ischemia), or may affect large areas of brain tissue (global ischemia). Significant brain injury can occur after the immediate ischemic event. Neuronal death and injury after cerebral ischemia involve pathological changes associated with necrosis and delayed apoptosis. Neurons in the infarction core of focal, severe stroke are immediately dead and cannot be saved by pharmacologic intervention. The ischemic penumbra, consisting of the brain tissue around the core in focal ischemic stroke, and the sensitive neurons/network in global cerebral ischemia, however, are maintained by a diminished blood supply. The damage to this penumbral brain tissue occurs in a “delayed” manner, starting 4-6 hours as the second phase or days and weeks later as the so-called third phase, after ischemic stroke.

A consistent consequence of cerebral ischemia/hypoxia in humans and other mammals is central nervous system dysfunction, the nature of which depends on the location and extent of injury. Global cerebral ischemia/hypoxia selectively injures or damages the pyramidal neurons in the dorsal hippocampal CA1 area, which are essential for episodic memory, providing a sensitive measure for monitoring ischemic damage and recovery functionally. After a cerebral ischemia of about 15 minutes, for example, the hippocampal CA1 pyramidal cells start to degenerate within 2-3 days, and reach the maximal extent of cell death a week after the ischemic event. The sensitive neuronal structures in global cerebral ischemia and the ischemic penumbra are “at-risk” tissues. They can be salvage through intervention and further damage limited in the subsequent days or weeks thereafter, which determine dramatic differences in long-term disability.

Following ischemic stroke, there is a transient loss of blood-brain barrier (BBB) function that happens within minutes or hours of the event as the interruption in blood flow and lack of oxygen leads to increased BBB permeability. DiNapoli et al., Neurobiology of Aging (2008) vol. 29, pp. 753-764. Disruption of the BBB, in turn, results in loss of ionic homeostasis and loss of neurotransmitter homeostasis. Immune cells and toxic compounds can enter the brain during that period, providing an added neurotoxic insult. Edema can form during the early stages of ischemia with a rate related to the rate of sodium transport from blood to brain, i.e., increased sodium transport across the BBB contributes to cerebral edema formation. Betz and Coester, Stroke (1990), vol. 21, pp. 1199-1204. Thus, measurements of both edema and ion uptake in the brain are indicators of brain pathology following stroke. The loss of the integrity of the barrier may lead to adverse hemorrhages as a consequence of thrombolytic therapy, e.g., administration of recombinant tissue plasminogen activator (rTPA). Tanne et al., Nature Reviews Neurology (2008), vol. 4, pp. 644-645.

Despite the medical emergency presented by stroke, and preclinical studies suggesting agents that may be effective in arresting the pathological processes involved, options for treating stroke remain limited. The main treatment available is rTPA, a thrombolytic agent and the only drug currently approved by the U.S. Food and Drug Administration for acute/urgent treatment of ischemic stroke. The rTPA protein is an enzyme (serine protease) that initiates local fibrinolysis via fibrin-enhanced conversion of plasminogen to plasmin. rTPA is used to improve neurologic recovery and reduce the incidence of disability. Experimental models of stroke use rTPA, for example, in reperfusion after inducing focal embolic ischemia via middle cerebral artery occlusion (MCAO). DiNapoli et al., J. Neurosci Methods (2006), vol. 154, pp. 233-238.

The effectiveness of rTPA and other potential agents for arresting infarct development depends on early administration or even before the ischemic event, if possible. Treatment with rTPA is designed to achieve early arterial recanalization such that rTPA must be administered within 3 hours after the event to be effective. This time dependency limits its clinical usefulness; the narrow therapeutic time window and exclusion criteria in treating ischemic stroke leads to about only 5% of candidate patients receiving effective intravenous thrombolytic therapy. For example, one study reported 13% mortality at 30 days after an acute ischemic stroke, with more than two thirds of the deaths related to the initial stroke. Nedeltcheva et al., Swiss Med. Wkly (2010), vol. 140, pp. 254-259. The recommended dose of rTPA is 0.9 mg/kg (maximum dose 90 mg) where 10% is given by rapid (˜1 min.) IV injection and the remainder by constant infusion over 60 min. No aspirin, heparin, or warfarin should be administered for 24 hours following rTPA. rTPA is sold under the names alteplase (Activase®) and streptokinase (Streptase®).

Use of rTPA following stroke is controversial because it carries an increased risk of intracranial hemorrhage, reperfusion injury, and diminishing cerebral artery reactivity. Thus, rTPA is should not be administered to treat hemorrhagic stroke. Unfortunately, it may not be immediately apparent whether a patient suffered an ischemic or hemorrhagic stroke, which further limits the usefulness of rTPA within its limited therapeutic time window. In addition, hemorrhagic transformation can spontaneously follow ischemic stroke. For example, one study found that 6.4% of patients with large strokes developed substantial brain hemorrhage as a complication from being given rTPA. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, N. Engl. J. Med. (1995), vol. 333, pp. 1581-1587.

rTPA is contraindicated or advised against in the following patient populations:

-   -   Evidence of intracranial hemorrhage on pretreatment CT scan     -   Clinical presentation suggestive of subarachnoid hemorrhage,         even with normal CT scan     -   Active internal bleeding     -   Known bleeding diathesis, including but not limited to: having a         platelet count <100,000/mm; receiving heparin within 48 hours         and having an elevated activated partial thromboplastin (aPTT)         greater than upper limit of normal for laboratory; and current         use of oral anticoagulants (e.g., warfarin sodium) or recent use         with an elevated prothrombin time>15 seconds     -   Within 3 months any intracranial surgery, serious head trauma,         or previous stroke     -   History of gastrointestinal or urinary tract hemorrhage within         21 days     -   Recent arterial puncture at a noncompressible site     -   Recent lumbar puncture     -   On repeated measurements, systolic blood pressure greater than         185 mm Hg or diastolic blood pressure greater than 110 mm Hg at         the time treatment is to begin, and patients requiring         aggressive treatment to reduce blood pressure to within these         limits.     -   History of intracranial hemorrhage     -   Abnormal blood glucose (<50 mg/dL or >400 mg/dL)     -   Post myocardial infarction pericarditis     -   Patient observed to have seizure at the same time the onset of         stroke symptoms were observed     -   Known arteriovenous malformation, or aneurysm         See, e.g., TPA Stroke Study Group Guidelines, The Brian Attack         Coalition (available at         http://www.stroke-site.org/guidelines/tpa_guidelines.html).

Studies suggest an association between hematocrit, reduced reperfusion and greater infarct size, and between elevated hemoglobin levels and increased rates of all-cause death. Tanne et al., BMC Neurology (2010), vol. 10:22, pp. 1-7. Elevated levels of glycated hemoglobin (HbA1c) increases the risk of heart attacks and strokes in diabetic patients. Glycated hemoglobin, even at levels considered in the normal range, can also be an independent predictor of ischemic stroke in non-diabetic adults. Selvin et al., N. Engl. J. Med. (2010), vol. 362, pp. 800-811. Elevated hemoglobin may also increase the risk of stroke in patients with chronic kidney disease.

Low hemoglobin levels (e.g., levels >6.0% or 8.8 g/dL, anemia) have also been identified as a risk factor for ischemic stroke, especially following cardiac surgery. In addition, anemia can worsen brain ischemia following acute ischemic stroke, and is associated with a poor prognosis and increased mortality after one year compared with non-anemic stroke patients (hemoglobin <13 g/dL in males, <12 g/dL in women). Tanne et al., BMC Neurology (2010), 10:22. Studies have also reported that children with sickle cell anemia have an increased stroke risk.

Protein Kinase C

Protein kinase C (PKC) is one of the largest gene families of non-receptor serine-threonine protein kinases. Since the discovery of PKC in the early eighties and its identification as a major receptor for phorbol esters, a multitude of physiological signaling mechanisms have been ascribed to this enzyme. Kikkawa et al., J. Biol. Chem. (1982), vol. 257, pp. 13341-13348; Ashendel et al., Cancer Res. (1983), vol. 43: 4333-4337. The interest in PKC stems from its unique ability to be activated in vitro by calcium and diacylglycerol (and phorbol ester mimetics), an effector whose formation is coupled to phospholipid turnover by the action of growth and differentiation factors. Activation of PKC involves binding of 1,2-diacylglycerol (DAG) and/or 1,2-diacyl-sn-glycero-3-phospho-L-serine (phosphatidyl-L-serine, PS) at different binding sites. An alternative approach to activating PKC directly is through indirect PKC activation, e.g., by activating phospholipases such as phospholipase Cy, by stimulating the Ser/Thr kinase Akt by way of phosphatidylinositol 3-kinase (P13K), or by increasing the levels of DAG, the endogenous activator. Nelson et al., Trends in Biochem. Sci. (2009) vol. 34, pp. 136-145. Diacylglycerol kinase inhibitors, for example, may enhance the levels of the endogenous ligand diacylglycerol, thereby producing activation of PKC. Meinhardt et al., Anti-Cancer Drugs (2002), vol. 13, pp. 725-733. Phorbol esters are not suitable compounds for eventual drug development because of their tumor promotion activity. Ibarreta et al. Neuroreport (1999), vol. 10, pp. 1035-1040).

The PKC gene family consists of 11 genes, which are divided into four subgroups: (1) classical PKC α, β1, β2 ((β1 and β2 are alternatively spliced forms of the same gene) and γ; (2) novel PKC δ, ε, η, and θ; (3) atypical PKC ζ and ι/λ; and (4) PKC μ. PKC μ resembles the novel PKC isoforms but differs by having a putative transmembrane domain. Blobe et al. Cancer Metastasis Rev. (1994), vol. 13, pp. 411-431; Hug et al. Biochem. J. (1993) vol. 291, pp. 329-343; Kikkawa et al. Ann. Rev. Biochem. (1989), vol. 58, pp. 31-44. The classical PKC isoforms α, β1, β2, and γ are Ca²⁺, phospholipid, and diacylglycerol-dependent, whereas the other isoforms are activated by phospholipid, diacylglycerol but are not dependent on Ca²⁺ and no activator may be necessary. All isoforms encompass 5 variable (V1-V5) regions, and the α,β, and γ isoforms contain four (C1-C4) structural domains which are highly conserved. All isoforms except PKC α, β, and γ lack the C2 domain, the ι/λ and η isoforms also lack nine of two cysteine-rich zinc finger domains in C1 to which diacylglycerol binds. The C1 domain also contains the pseudosubstrate sequence which is highly conserved among all isoforms, and which serves an autoregulatory function by blocking the substrate-binding site to produce an inactive conformation of the enzyme. House et al., Science (1987), vol. 238, pp. 1726-1728.

Because of these structural features, diverse PKC isoforms are thought to have highly specialized roles in signal transduction in response to physiological stimuli as well as in neoplastic transformation and differentiation. Nishizuka, Cancer (1989), vol. 10, pp. 1892-1903; Glazer, pp. 171-198 in Protein Kinase C, I. F. Kuo, ed., Oxford U. Press, 1994. For a discussion of PKC modulators see, for example, International Application No. PCT/US97/08141 (WO 97/43268) and U.S. Pat. Nos. 5,652,232; 6,080,784; 5,891,906; 5,962,498; 5,955,501; 5,891,870 and 5,962,504, each incorporated by reference herein in its entirety.

The activation of PKC has been shown to improve learning and memory. See, e.g., Hongpaisan et al., Proc. Natl. Acad. Sci. (2007) vol. 104, pp. 19571-19578; International Application Nos. PCT/US2003/007101 (WO 2003/075850); PCT/US2003/020820 (WO 2004/004641); PCT/US2005/028522 (WO 2006/031337); PCT/US2006/029110 (WO 2007/016202); PCT/US2007/002454 (WO 2008/013573); PCT/US2008/001755 (WO 2008/100449); PCT/US2008/006158 (WO 2008/143880); PCT/US2009/051927 (WO 2010/014585); and PCT/US2011/000315; and U.S. application Ser. Nos. 12/068,732; 10/167,491 (now U.S. Pat. No. 6,825,229); 12/851,222; 11/802,723; 12/068,742; and 12/510,681; each incorporated by reference herein in its entirety. PKC activators have been used to treat memory and learning deficits induced by stroke upon administration 24 hours or more after inducing global cerebral ischemia through two-vessel occlusion combined with a short term (˜14 minutes) systemic hypoxia. Sun et al., Proc. Natl. Acad. Sci. (2008) vol. 105, pp. 13620-13625; Sun et al., Proc. Natl. Acad. Sci. (2009) vol. 106, pp. 14676-14680.

The present disclosure relates to a method of treating stroke in a subject who has suffered an ischemic event comprising administering to the subject an anticoagulant and a protein kinase C (PKC) activator.

The present disclosure further provides for a composition comprising a therapeutically effective amount of a protein kinase C (PKC) activator and a therapeutically effective amount of an anticoagulant.

The present disclosure further provides for a kit comprising a composition comprising an anticoagulant and a composition comprising a protein kinase C (PKC) activator.

Also disclosed herein is a method of treating stroke in a subject comprising: (a) identifying a subject having suffered a stroke; (b) administering to the subject a therapeutically-effective amount of a protein kinase C (PKC) activator; (c) determining whether the subject suffered an ischemic stroke or hemorrhagic stroke; and (d) if the subject suffered an ischemic stroke, administering a therapeutically-effective amount of an anticoagulant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a the amount of hemoglobin the ipsilateral and contralateral cortices following ischemic stroke in rats treated with either rTPA at 6 hours following stroke, or a combination of bryostatin-1 administered 2 hours after the stroke, followed 6 hours later by rTPA.

FIG. 2 shows a the percent of brain edema following ischemic stroke in rats treated with either rTPA at 6 hours following the stroke, or a combination of bryostatin-1 administered 2 hours after the stroke, followed 6 hours later by rTPA.

FIG. 3 shows the results of uptake of Evans Blue dye in the ipsilateral and contralateral cortices in rats treated with rTPA 2 hours following the stroke, or a combination of rTPA at 2 hours followed 6 hours later with bryostatin-1.

FIG. 4 shows the results of sodium fluoride uptake in the ipsilateral and contralateral cortices in rats treated with rTPA 2 hours following the stroke, or a combination of rTPA at 2 hours followed 6 hours later with bryostatin-1.

DETAILED DESCRIPTION

Particular aspects of the disclosure are described in greater detail below. The terms and definitions as used in the present application and as clarified herein are intended to represent the meaning within the present disclosure. The patent and scientific literature referred to herein is hereby incorporated by reference. The terms and definitions provided herein control, if in conflict with terms and/or definitions incorporated by reference.

The singular forms “a,” “an,” and “the” include plural reference unless the context dictates otherwise.

The terms “approximately” and “about” mean to be nearly the same as a referenced number or value including an acceptable degree of error for the quantity measured given the nature or precision of the measurements. As used herein, the terms “approximately” and “about” should be generally understood to encompass ±20% of a specified amount, frequency or value. Numerical quantities given herein are approximate unless stated otherwise, meaning that term “about” or “approximately” can be inferred when not expressly stated.

The terms “administer,” “administration,” or “administering” as used herein refer to (1) providing, giving, dosing and/or prescribing by either a health practitioner or his authorized agent or under his direction a composition according to the disclosure, and (2) putting into, taking or consuming by the patient or person himself or herself, a composition according to the disclosure. As used herein, “administration” of a composition includes any route of administration, including oral, intravenous, subcutaneous, intraperitoneal, and intramuscular.

As used herein, the term “subject” means a mammal, i.e., a human or a non-human mammal.

The phrase “a therapeutically effective amount” refers to an amount of a therapeutic agent that results in a measurable therapeutic response. A therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy, including improvement of symptoms and surrogate clinical markers. Thus, a therapeutic response will generally be an amelioration or inhibition of one or more symptoms of a disease or condition, e.g., stroke. A measurable therapeutic response also includes a finding that a symptom or disease is prevented or has a delayed onset, or is otherwise attenuated by the therapeutic agent. thus, a “therapeutically effective amount” as used herein refers to an amount sufficient to reduce one or more symptom(s) or condition(s) associated with stroke including but not limited to hemorrhagic transformation, disruption of the blood-brain barrier, increase in hemoglobin levels, and mortality.

As used herein, “protein kinase C activator” or “PKC activator” means a substance that increases the rate of the reaction catalyzed by protein kinase C by binding to the protein kinase C.

As used herein “macrocyclic lactone” refers to a compound comprising a macrolide ring, i.e., a large macrocyclic lactone ring to which one or more deoxy sugars may be attached.

The term “neurodegeneration” refers to the progressive loss of structure or function of neurons, including death of neurons.

The term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject.

While the present disclosure generally describes use of rTPA, other anticoagulants and anticoagulant therapies suitable for the treatment of stroke are also contemplated. Further, it is understood that the present disclosure is not limited to a specific manufactured type of TPA (e.g., rTPA), but includes TPA generally.

The present disclosure generally relates to compositions, kits, and methods of use of an anticoagulant, e.g., rTPA and a PKC activator to treat stroke. In some embodiments, the administration of a PKC activator may extend the time that rTPA can be administered after a stroke (e.g., after an ischemic event) while still retaining efficacy. Further, the combination of rTPA and a PKC activator may reduce mortality, reduce hemorrhagic transformation, and/or reduce disruptions to the blood-brain barrier (BBB) caused by stroke. In addition, the administration of rTPA and a PKC activator may reduce the level of assayed hemoglobin, wherein elevated hemoglobin is a risk factor for reduced reperfusion, greater infarct size, and/or mortality due to stroke.

Sliding Temporal Window

The present disclosure encompasses “sliding temporal windows” for administration of a PKC activator and rTPA to a stroke victim. In the methods presently disclosed, a PKC activator may be administered before, after, and/or in combination with rTPA. In some embodiments of the present disclosure, rTPA and a PKC activator are administered at the same time. Thus, the present disclosure contemplates “sliding temporal windows” for administration of a PKC activator and rTPA to a subject. The term “sliding temporal window” refers to the notion that a PKC activator and rTPA can be administered in any order to a subject that has suffered a stroke, at any time relative to one another, and at any time relative to when the stroke occurred.

At least four scenarios are contemplated:

Scenario 1: In some embodiments of the present disclosure, a PKC activator may be administered to a subject one or more times within a given time period after having suffered a stroke, followed by administration of rTPA one or more times after another time period. The PKC activator may be administered at any time after the occurrence of a stroke, generally within about 24 hours. For example, the PKC activator may be administered to a subject about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after a stroke. The rTPA may then be administered to the subject after the PKC activator, e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after administration of the PKC activator.

For example, in some embodiments, the PKC activator is administered within 24 hours after the ischemic event, such as from about 1 hour to about 12 hours or from about 2 hours to about 6 hours after the ischemic event. rTPA is then administered within 24 hours after administration of the PKC activator, such as from about 1 hour to about 12 hours or from about 2 hours to about 6 hours after administration of the PKC activator. In one embodiment, the PKC activator is administered within about 6 hours after the ischemic event and the rTPA is administered within about 2 hours after administration of the PKC activator. In another embodiment, the PKC activator is administered about 3 hours after the ischemic event and the rTPA is administered about 2 hours after the PKC activator.

Scenario 2: In some embodiments, rTPA may be administered to a subject one or more times within a given time period after having suffered a stroke, followed by administration of a PKC activator one or more times after another time period. The rTPA may be administered at any time after the occurrence of a stroke, generally within about 24 hours. For example, the rTPA may be administered to a subject about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after a stroke. The PKC activator may then be administered to the subject after the rTPA, e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after administration of the rTPA.

For example, in some embodiments, the rTPA is administered within 24 hours after the ischemic event, such as from about 1 hour to about 12 hours or from about 2 hours to about 6 hours after the ischemic event. The PKC activator is then administered within 24 hours after administration of the rTPA, such as from about 1 hour to about 12 hours or from about 2 hours to about 6 hours after the rTPA. In one embodiment, rTPA is administered within about 6 hours after the ischemic event and the PKC activator is administered within about 2 hours after the rTPA. In another embodiment, rTPA is administered about 3 hours after the ischemic event and the PKC activator is administered about 2 hours after the rTPA.

Scenario 3: In other embodiments of the present disclosure, a PKC activator may be administered to a subject one or more times within a given time period after having suffered a stroke, followed by rTPA one or more times after another time period, and further followed by administration of a PKC activator one or more times a period of time later. For example, the PKC activator may be administered to a subject about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after a stroke. The rTPA may then be administered to the subject about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after administration of the PKC activator. Thereafter, another PKC activator may be administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after administration of the rTPA. The PKC activator administered before and after the rTPA may be the same or different.

Similarly, rTPA may be administered to a subject one or more times within a given time period after having suffered a stroke, followed by a PKC activator one or more times after another time period, and further followed by administration of the same or a different PKC activator one or more times a period of time later.

Scenario 4: In yet other embodiments, a PKC activator and rTPA may be administered at the same time to a subject after suffering a stroke. This may be done by directly administering a composition comprising a PKC activator and rTPA, or administering a composition comprising a PKC activator and a separate composition comprising rTPA in succession or rapid succession, one after the other in either order (i.e., the composition comprising a PKC activator may be administered first or the composition comprising rTPA may be administered first).

In some embodiments, the present disclosure provides a method for extending the therapeutic window for treating ischemic stroke with rTPA comprising administering a PKC activator before, after, or at the same time as rTPA. The recommended time period for administering rTPA (e.g., Activase®) is about 3 hours. In one embodiment of the present disclosure, for example, a PKC activator is administered to a subject about 2 hours after a stroke followed by administration of rTPA about 6 hours later (i.e., about 8 hours after the stroke). In another embodiment, rTPA is administered to a subject about 6 hours after a stroke followed by administration of a PKC activator about 2 hours later (i.e., about 8 hours after the stroke).

At least one embodiment of the present disclosure provides for treatment of a subject who has suffered a stroke before it is known whether the subject suffered an ischemic stroke or a hemorrhagic stroke. For example, the present disclosure provides for a method of identifying a subject who has suffered a stroke, administering a therapeutically-effective amount of a PKC activator, and determining whether the subject suffered an ischemic stroke or a hemorrhagic stroke. The determination regarding the type of stroke suffered may be made by any suitable means known in the medical arts including, for example, a computed tomography (CT) scan. If the subject suffered an ischemic stroke, a therapeutically-effective amount of rTPA may be administered. If the subject suffered a hemorrhagic stroke, however, rTPA is not administered. Thus, in some embodiments of the present disclosure, extending the therapeutic time window for treating stroke with rTPA allows for a determination of whether a subject suffered an ischemic stroke or a hemorrhagic stroke.

PKC Activators

PKC activators suitable for the methods, compositions, and kits disclosed herein include, for example, macrocyclic lactones, e.g., bryostatin and neristatin classes, that act to stimulate PKC. Of the bryostatin class of compounds, bryostatin-1 has been shown to activate PKC without tumor promotion. Bryostatin-1 may be particularly useful as a PKC activator because the dose response curve is biphasic and bryostatin-1 demonstrates differential regulation of PKC isozymes including PKCα, PKCδ and PKCε. Bryostatin-1 has undergone toxicity and safety studies in animals and humans, and is actively investigated as an anti-cancer agent.

Macrocyclic lactones generally comprise 14-, 15-, or 16-membered lactone rings. Macrolides belong to the polyketide class of natural products. Macrocyclic lactones and derivatives thereof are described, for example, in U.S. Pat. Nos. 6,187,568; 6,043,270; 5,393,897; 5,072,004; 5,196,447; 4,833,257; and 4,611,066; and 4,560,774; each incorporated by reference herein in its entirety. Those patents describe various compounds and various uses for macrocyclic lactones including their use as an anti-inflammatory or anti-tumor agent. Szallasi et al. J. Biol. Chem. (1994), vol. 269, pp. 2118-2124; Zhang et al., Cancer Res. (1996), vol. 56, pp. 802-808; Hennings et al. Carcinogenesis (1987), vol. 8, pp. 1343-1346; Varterasian et al. Clin. Cancer Res. (2000), vol. 6, pp. 825-828; Mutter et al. Bioorganic & Med. Chem. (2000), vol. 8, pp. 1841-1860; each incorporated by reference herein in its entirety. The bryostatin and neristatin compounds were originally isolated from the marine bryozoan Bugula neritina L.

In one embodiment of the present disclosure, the PKC activator is a macrocyclic lactone such as a bryostatin or neristatin. Bryostatins include, for example, bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin-10, bryostatin-11, bryostatin-12, bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16, bryostatin-17, and bryostatin-18. In at least one embodiment, the bryostatin is bryostatin-1. Neristatins suitable for the present disclosure include, for example, neristatin-1.

Analogs of bryostatin, commonly referred to as bryologs, are one particular class of PKC activators that are suitable for use in the present disclosure. Table 1 summarizes structural characteristics of several bryologs and demonstrates variability in their affinity for PKC (ranging from 0.25 nM to 10 μM). Structurally, they are all similar. While bryostatin-1 has two pyran rings and one 6-membered cyclic acetal, in most bryologs one of the pyrans of bryostatin-1 is replaced with a second 6-membered acetal ring. This modification reduces the stability of bryologs, relative to bryostatin-1, for example, in both strong acid or base, but has little significance at physiological pH. Bryologs also have a lower molecular weight (ranging from about 600 g/mol to 755 g/mol), as compared to bryostatin-1 (988), a property which facilitates transport across the blood-brain barrier.

TABLE 1 Bryologs. PKC Affin Name (nM) MW Description Bryostatin-1 1.35 988 2 pyran + 1 cyclic acetal + macrocycle Analog 1 0.25 737 1 pyran + 2 cyclic acetal + macrocycle Analog 2 6.50 723 1 pyran + 2 cyclic acetal + macrocycle Analog 7a — 642 1 pyran + 2 cyclic acetals + macrocycle Analog 7b 297 711 1 pyran + 2 cyclic acetals + macrocycle Analog 7c 3.4 726 1 pyran + 2 cyclic acetals + macrocycle Analog 7d 10000 745 1 pyran + 2 cyclic acetals + macrocycle, acetylated Analog 8 8.3 754 2 cyclic acetals + macrocycle Analog 9 10000 599 2 cyclic acetals

Analog 1 exhibits the highest affinity for PKC. Wender et al., Curr. Drug Discov. Technol. (2004), vol. 1, pp. 1-11; Wender et al. Proc. Natl. Acad. Sci. (1998), vol. 95, pp. 6624-6629; Wender et al., J. Am. Chem. Soc. (2002), vol. 124, pp. 13648-13649, each incorporated by reference herein in their entireties. Only Analog 1 exhibits a higher affinity for PKC than bryostatin. Analog 2, which lacks the A ring of bryostatin-1, is the simplest analog that maintains high affinity for PKC. In addition to the active bryologs, Analog 7d, which is acetylated at position 26, has virtually no affinity for PKC.

B-ring bryologs may also be used in the present disclosure. These synthetic bryologs have affinities in the low nanomolar range. Wender et al., Org Lett. (2006), vol. 8, pp. 5299-5302, incorporated by reference herein in its entirety. B-ring bryologs have the advantage of being completely synthetic, and do not require purification from a natural source.

A third class of suitable bryostatin analogs is the A-ring bryologs. These bryologs have slightly lower affinity for PKC than bryostatin-1 (6.5 nM, 2.3 nM, and 1.9 nM for bryologs 3, 4, and 5, respectively) and a lower molecular weight.

Bryostatin analogs are described, for example, in U.S. Pat. Nos. 6,624,189 and 7,256,286.

A number of derivatives of diacylglycerol (DAG) bind to and activate PKC. Niedel et al., Proc. Natl. Acad. Sci. (1983), vol. 80, pp. 36-40; Mori et al., J. Biochem. (1982), vol. 91, pp. 427-431; Kaibuchi et al., J. Biol. Chem. (1983), vol. 258, pp. 6701-6704. However, DAG and DAG derivatives are of limited value as drugs. Activation of PKC by diacylglycerols is transient, because they are rapidly metabolized by diacylglycerol kinase and lipase. Bishop et al. J. Biol. Chem. (1986), vol. 261, pp. 6993-7000; Chuang et al. Am. J. Physiol. (1993), vol. 265, pp. C927-C933; incorporated by reference herein in their entireties. The fatty acid substitution determines the strength of activation. Diacylglycerols having an unsaturated fatty acid are most active. The stereoisomeric configuration is important; fatty acids with a 1,2-sn configuration are active while 2,3-sn-diacylglycerols and 1,3-diacylglycerols do not bind to PKC. Cis-unsaturated fatty acids may be synergistic with diacylglycerols. In at least one embodiment, the term “PKC activator” expressly excludes DAG or DAG derivatives.

Isoprenoids are PKC activators also suitable for the present disclosure. Farnesyl thiotriazole, for example, is a synthetic isoprenoid that activates PKC with a K_(d) of 2.5 μM. Farnesyl thiotriazole, for example, is equipotent with dioleoylglycerol, but does not possess hydrolyzable esters of fatty acids. Gilbert et al., Biochemistry (1995), vol. 34, pp. 3916-3920; incorporated by reference herein in its entirety. Farnesyl thiotriazole and related compounds represent a stable, persistent PKC activator. Because of its low molecular weight (305.5 g/mol) and absence of charged groups, farnesyl thiotriazole would be expected to readily cross the blood-brain barrier.

Octylindolactam V is a non-phorbol protein kinase C activator related to teleocidin. The advantages of octylindolactam V (specifically the (−)-enantiomer) include greater metabolic stability, high potency (EC₅₀=29 nM) and low molecular weight that facilitates transport across the blood brain barrier. Fujiki et al. Adv. Cancer Res. (1987), vol. 49 pp. 223-264; Collins et al. Biochem. Biophys. Res. Commun. (1982), vol. 104, pp. 1159-4166, each incorporated by reference herein in its entirety.

Gnidimacrin is a daphnane-type diterpene that displays potent antitumor activity at concentrations of 0.1 nM-1 nM against murine leukemias and solid tumors. It acts as a PKC activator at a concentration of 0.3 nM in K562 cells, and regulates cell cycle progression at the G1/S phase through the suppression of Cdc25A and subsequent inhibition of cyclin dependent kinase 2 (Cdk2) (100% inhibition achieved at 5 ng/ml). Gnidimacrin is a heterocyclic natural product similar to bryostatin, but somewhat smaller (MW=774.9 g/mol).

Iripallidal is a bicyclic triterpenoid isolated from Iris pallida. Iripallidal displays anti-proliferative activity in a NCI 60 cell line screen with GI₅₀ (concentration required to inhibit growth by 50%) values from micromolar to nanomolar range. It binds to PKCa with high affinity (K_(i)=75.6 nM). It induces phosphorylation of Erk½ in a RasGRP3-dependent manner. Its molecular weight is 486.7 g/mol. Iripallidal is about half the size of bryostatin and lacks charged groups.

Ingenol is a diterpenoid related to phorbol but less toxic. It is derived from the milkweed plant Euphorbia peplus. Ingenol 3,20-dibenzoate, for example, competes with [3H] phorbol dibutyrate for binding to PKC (K_(i)=240 nM). Winkler et al., J. Org. Chem. (1995), vol. 60, pp. 1381-1390, incorporated by reference herein. Ingenol-3-angelate exhibits antitumor activity against squamous cell carcinoma and melanoma when used topically. Ogbourne et al. Anticancer Drugs (2007), vol. 18, pp. 357-362, incorporated by reference herein.

Napthalenesulfonamides, including N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, are members of another class of PKC activators. SC-10 activates PKC in a calcium-dependent manner, using a mechanism similar to that of phosphatidylserine. Ito et al., Biochemistry (1986), vol. 25, pp. 4179-4184, incorporated by reference herein. Naphthalenesulfonamides act by a different mechanism than bryostatin and may show a synergistic effect with bryostatin or member of another class of PKC activators. Structurally, naphthalenesulfonamides are similar to the calmodulin (CaM) antagonist W-7, but are reported to have no effect on CaM kinase.

Diacylglycerol kinase inhibitors may also be suitable as PKC activators in the present disclosure by indirectly activating PKC. Examples of diacylglycerol kinase inhibitors include, but are not limited to, 6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [34244-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (R59949).

A variety of growth factors, such as fibroblast growth factor 18 (FGF-18) and insulin growth factor, function through the PKC pathway. FGF-18 expression is up-regulated in learning, and receptors for insulin growth factor have been implicated in learning. Activation of the PKC signaling pathway by these or other growth factors offers an additional potential means of activating PKC.

Growth factor activators, including 4-methyl catechol derivatives like 4-methylcatechol acetic acid (MCBA) that stimulate the synthesis and/or activation of growth factors such as NGF and BDNF, also activate PKC as well as convergent pathways responsible for synaptogenesis and/or neuritic branching.

In some embodiments of the present disclosure, administering a PKC activator and rTPA may reduce mortality in a subject 24 hours after stroke. For example, mortality after 24 hours may be reduced by at least 20%, at least 30%, at least 40%, or at least 50%. In at least one embodiment, administering a PKC activator and rTPA reduces mortality 24 hours after stroke by at least 40%.

In some embodiments, the combination of a PKC activator and rTPA may reduce disruption of the blood-brain barrier after stroke. The combination of a PKC activator and rTPA may also reduce hemorrhagic transformation. In some embodiments, for example, administering a PKC activator and rTPA after a stroke reduces hemoglobin levels, wherein a reduction in hemoglobin indicates a reduction in hemorrhagic transformation and/or a reduction in disruption of the blood-brain barrier. In some embodiments, the hemoglovin level is reduced by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. In at least one embodiment, for example, administering a PKC activator and rTPA after stroke reduces hemoglobin levels by about 50%. Reduced disruption of the blood-brain barrier may also be assessed by measuring extravasation of albumin. DiNapoli et al., Neurobiology of Aging (2008), vol. 29, pp. 753-764.

In some embodiments, administering a PKC activator and rTPA may limit the size of the infarction due to stroke, e.g., limit the tissue damage caused by an ischemic event.

Formulation and Administration

The formulations of the pharmaceutical compositions described herein may be prepared by any suitable method known in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with a carrier or one or more other accessory ingredients, then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, and other mammals.

In some embodiments, the PKC activator and anticoagulant, e.g., rTPA, are formulated together. In other embodiments, the PKC activator and rTPA are formulated separately.

The compositions disclosed herein may be administrated by any suitable route including oral, parenteral, transmucosal, intranasal, inhalation, or transdermal routes. Parenteral routes include intravenous, intra-arteriolar, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal, and intracranial administration. A suitable route of administration may be chosen to permit crossing the blood-brain barrier. Rapoport et al., J. Lipid Res. (2001) vol. 42, pp. 678-685.

The compositions disclosed herein may be formulated according to conventional methods, and may include any pharmaceutically acceptable additives, such as excipients, lubricants, diluents, flavorants, colorants, buffers, and disintegrants. See e.g., Remington's Pharmaceutical Sciences, 20^(th) Ed., Mack Publishing Co. 2000.

In some embodiments, the PKC activator is formulated in a solid oral dosage form. For oral administration, the composition may take the form of a tablet or capsule prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods generally known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-phydroxybenzoates or sorbic acid). The preparations may also comprise buffer salts, flavoring, coloring and sweetening agents as appropriate.

In other embodiments of the present disclosure, the PKC activator may be formulated for parenteral administration such as bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, dispersions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

In some embodiments, the PKC activator may be formulated with a pharmaceutically-acceptable carrier for administration. Pharmaceutically acceptable carriers include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are generally known in the art and may be described, for example, in Remington's Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, Pa., 1985, incorporated by reference herein.

In some embodiments, the PKC activator may be formulated with a hydrophobic carrier for administration. Hydrophobic carriers include inclusion complexes, dispersions (such as micelles, microemulsions, and emulsions), and liposomes. Exemplary hydrophobic carriers include inclusion complexes, micelles, and liposomes. See, e.g., Remington's: The Science and Practice of Pharmacy 20th ed., ed. Gennaro, Lippincott: Philadelphia, Pa. 2003. The PKC activators presently disclosed may be incorporated into hydrophobic carriers, for example as at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the total carrier by weight. In addition, other compounds may be included either in the hydrophobic carrier or the solution, e.g., to stabilize the formulation.

In some embodiments, the PKC activator may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the PKC activator may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

In another embodiment, the PKC activator may be delivered in a vesicle, such as a micelle, liposome, or an artificial low-density lipoprotein (LDL) particle. See, e.g., U.S. Pat. No. 7,682,627.

The doses for administration may suitably be prepared so as to deliver from about 1 mg to about 10 g, such as from about 10 mg to about 1 g, or for example, from about 250 mg to about 500 mg of the PKC activator per day. When prepared for topical administration or parenteral formulations they may be made in formulae containing from about 0.01% to about 60% by weight of the final formulation, such as from about 0.1% to about 30% by weight, such as from about 1% to about 10% by weight. A suitable dose can be determined by methods known in the art and according to clinically relevant factors such as the age of the patient.

In at least one embodiment, the PKC activator is formulated for intravenous administration. The PKC activator may be formulated for intravenous administration of a dose ranging from about 25 μg/m² to about 50 μg/m². In some embodiments, the PKC activator and rTPA are both formulated for intravenous administration. The rTPA may be formulated for intravenous administration of a dose of about 0.9 mg/kg. The PKC and rTPA may be formulated together for intravenous administration, or they may be formulated separately for intravenous administration.

Kits

The present disclosure further relates to kits that may be utilized for preparing and administering pharmaceutical compositions of an anticoagulant, e.g., rTPA, and a PKC activator disclosed herein to a subject in need thereof. The kits may also comprise devices such as syringes for administration of the pharmaceutical compositions described herein.

In some embodiments, the kits may comprise one or more vials, syringes, needles, ampules, cartridges, bottles or other such vessels for storing and/or subsequently mixing compositions of rTPA and PKC activator disclosed herein. In certain embodiments, the devices, syringes, ampules, cartridges, bottles or other such vessels for storing and/or subsequently mixing the compositions of rTPA and a PKC activator disclosed herein may, or may not have more than one chamber.

In still further embodiments, the compositions of rTPA and a PKC activator disclosed herein may be stored in one or more graduated vessels (such as a syringe or syringes or other device useful for measuring volumes).

In certain embodiments, the kits may comprise pharmaceutical compositions of rTPA and a PKC activator stored within the same or separate ampules, vials, syringes, cartridges, bottles or other such vessels.

The kits may also comprise one or more anesthetics, preferably local anesthetics. In certain embodiments, the anesthetics are in a ready-to-use formulation, such as, for example an injectable formulation (optionally in one or more pre-loaded syringes) or a formulation that may be applied topically to an area where the compositions of rTPA and PKC activator disclosed herein are to be administered.

Topical formulations of anesthetics may be in form an anesthetic applied to a pad, swab, towelette, disposable napkin, cloth, patch, bandage, gauze, cotton ball, Q-tip™, ointment, cream, gel, paste, liquid, or any other topically applied formulation. Anesthetics for use with the present invention may include, but are not limited to lidocaine, marcaine, cocaine and xylocaine, for example.

The kits may also contain instructions relating to the use of the pharmaceutical compositions of rTPA and a PKC activator and procedures for mixing, diluting or combining formulations of rTPA and a PKC activator. The instructions may also contain directions for properly diluting formulations of rTPA and/or a PKC activator to obtain a desired pH or range of pHs and/or a desired specific activity and/or protein concentration after mixing but prior to administration. The instructions may also contain dosing information. The instructions may also contain material directed to methods for selecting subjects for treatment with the disclosed pharmaceutical compositions of rTPA and a PKC activator. The kits may also include additional buffers, syringes, needles, needle-less injection devices, sterile pads or swabs.

The following examples are intended to illustrate the present disclosure without, however, being limiting in nature. It is understood that the skilled artisan will envision additional embodiments consistent with the disclosure provided herein.

EXAMPLES Example 1 Focal Ischemia Model of Stroke

A transient animal model of focal ischemia was used for these experiments. The middle cerebral artery (MCA) was surgically dissected and occluded in anesthetized rats by ligature, followed by reperfusion after a defined period (about 2 hours). Animal models transient ischemia via occlusion of the MCA (MCAO) are described in, e.g., Sicard and Fisher, Exp. & Transl. Stroke Med. (2009), vol. 1, pp. 1-7.

Example 2 Drug Administration

In a first experiment, rTPA was administered intravenously (˜0.9 mg/kg) 6 hours after the ischemic event, followed 2 hours later with a single intravenous administration of bryostatin-1 in a dosage range of from about 25 μg/m² to 50 μg/m².

In a second experiment, bryostatin-1 was administered intravenously (about 25 μg/m² to 50 μg/m²) 2 hours after the ischemic event, followed by intravenous administration of rTPA (˜0.9 mg/kg) about 6 hours later.

In a third experiment, rTPA was administered intravenously (˜0.9 mg/kg) 2 hours after the ischemic event, followed by intravenous administration of bryostatin-1 in a dosage range of from about 25 μg/m² to 50 μg/m² about 6 hours later.

Example 3 Results

1. Mortality

rTPA given 6 hours after the stroke, followed 2 hours later with bryostatin-1 led to 0% mortality 24 hours later (N=9 animals). In contrast, if rTPA was given 6 hours after the stroke, in the absence of subsequent treatment with bryostatin, 44% mortality was observed (N=6 animals).

2. Hemorrhage, Edema, and Blood-Brain Barrier Disruptions

Bryostatin-1 administered 2 hours after the stroke, followed 6 hours later by rTPA, resulted in a 50% reduction of assayed hemoglobin in the cortex and striatum, as compared to rTPA given 6 hours after the stroke without prior bryostatin-1 treatment (FIG. 1). Brain edema was also significantly reduced with this combination of rTPA and bryostatin-1 (FIG. 2).

The BBB permeability typically increase prior to the occurrence of edema following focal ischemia, such that edema can be used to measure BBB disruptions at the site of the ischemic lesion. In addition, the hemorrhage process is involved in the BBB disruption and edema. In one experiment, uptake of Evans Blue dye was used to measure BBB permeability, i.e., disruption, and hemorrhaging in ischemic animal models of stroke. FIG. 3 shows that combinations of bryostatin and administered according to the methods of the present disclosure significantly reduced uptake of Evans Blue dye in the ipsilateral and contralateral cortices.

Lastly, increased transport of sodium across the (BBB) contributes to cerebral edema formation in ischemic stroke. FIG. 4 shows that uptake of NaF in the ipsilateral and contralateral cortices is also reduced with the disclosed administration regimens of bryostatin-1 and rTPA.

The foregoing results demonstrate that the combination of bryostatin-1 with rTPA following ischemic stroke unexpectedly and significantly reduces mortality and brain injury following ischemic stroke. 

1. A method of treating a subject who has suffered an ischemic event comprising administering to the subject an anticoagulant and a protein kinase C (PKC) activator.
 2. The method of claim 1, wherein the anticoagulant is tissue plasminogen activator (TPA).
 3. The method of claim 1, wherein the PKC activator binds to the 1,2-diacylglycerol (DAG) site of PKC or indirectly activates PKC.
 4. The method of claim 1, wherein the PKC activator is chosen from macrocyclic lactones, diacylglycerol derivatives other than phorbol esters, isoprenoids, daphnane-type diterpenes, bicyclic triterpenoids, naphthalenesulfonamides, diacylglycerol kinase inhibitors, and growth factor activators.
 5. The method of claim 4, wherein the PKC activator is a macrocyclic lactone.
 6. The method of claim 5, wherein the macrocyclic lactone is chosen from bryostatin, bryologs, and neristatin.
 7. The method of claim 6, wherein the bryostatin is bryostatin-1.
 8. The method of claim 1, wherein the anticoagulant is administered before the PKC activator.
 9. The method of claim 8, wherein the anticoagulant is administered within 24 hours after the ischemic event.
 10. The method of claim 9, wherein the anticoagulant is administered from about 1 hour to about 12 hours after the ischemic event.
 11. The method of claim 10, wherein the anticoagulant is administered from about 2 hours to about 6 hours after the ischemic event.
 12. The method of claim 8, wherein the PKC activator is administered within 24 hours after administration of the anticoagulant.
 13. The method of claim 12, wherein the PKC activator is administered from about 1 hour to about 12 hours after administration of the anticoagulant.
 14. The method of claim 13, wherein the PKC activator is administered from about 2 hours to about 6 hours after the anticoagulant.
 15. The method of claim 8, wherein the anticoagulant is administered within about 6 hours after the ischemic event and the PKC activator is administered within about 2 hours after the anticoagulant.
 16. The method of claim 15, wherein the anticoagulant is administered about 3 hours after the ischemic event and the PKC activator is administered about 2 hours after the anticoagulant.
 17. The method of claim 1, wherein the PKC activator is administered before the anticoagulant.
 18. The method of claim 17, wherein the PKC activator is administered within 24 hours after the ischemic event.
 19. The method of claim 18, wherein the PKC activator is administered from about 1 hour to about 12 hours after the ischemic event.
 20. The method of claim 19, wherein the PKC activator is administered from about 2 hours to about 6 hours after the ischemic event.
 21. The method of claim 17, wherein the anticoagulant is administered within 24 hours after administration of the PKC activator.
 22. The method of claim 21, wherein the anticoagulant is administered from about 1 hour to about 12 hours after administration of the PKC activator.
 23. The method of claim 22, wherein the anticoagulant is administered from about 2 hours to about 6 hours after administration of the PKC activator.
 24. The method of claim 17, wherein the PKC activator is administered within about 6 hours after the ischemic event and the anticoagulant is administered within about 2 hours after administration of the PKC activator.
 25. The method of claim 24, wherein the PKC activator is administered about 3 hours after the ischemic event and the anticoagulant is administered about 2 hours after the PKC activator.
 26. The method of claim 1, wherein mortality is reduced with respect to administration of the anticoagulant alone.
 27. The method of claim 26, wherein mortality 24 hours after the stroke is reduced by at least 40%.
 28. The method of claim 1, wherein hemorrhagic transformation is reduced compared to administration of the anticoagulant alone.
 29. The method of claim 28, wherein the reduction in hemorrhagic transformation is determined by measuring a reduction in the subject's hemoglobin level.
 30. The method of claim 29, wherein the hemoglobin level is reduced by about 50%.
 31. The method of claim 1, wherein disruption of the blood-brain barrier is reduced compared to administration of the anticoagulant alone.
 32. A composition comprising a therapeutically effective amount of a protein kinase C (PKC) activator and a therapeutically effective amount of an anticoagulant.
 33. The composition of claim 32, wherein the anticoagulant is tissue plasmogin activator (TPA).
 34. The composition of claim 32, wherein the PKC activator binds to the 1,2-diacylglycerol (DAG) site of PKC or indirectly activates PKC.
 35. The composition of claim 32, wherein the PKC activator is chosen from macrocyclic lactones, diacylglycerol derivatives other than phorbol esters, isoprenoids, daphnane-type diterpenes, bicyclic triterpenoids, naphthalenesulfonamides, diacylglycerol kinase inhibitors, and growth factor activators.
 36. The composition of claim 35, wherein the PKC activator is a macrocyclic lactone.
 37. The composition of claim 36, wherein the macrocyclic lactone is chosen from bryostatin, bryologs, and neristatin.
 38. The composition of claim 37, wherein the bryostatin is bryostatin-1.
 39. A kit comprising a composition comprising an anticoagulant and a composition comprising a protein kinase C (PKC) activator.
 40. The kit of claim 39, wherein the anticoagulant is tissue plasmogin activator (TPA).
 41. The kit of claim 39, wherein the PKC activator binds to the 1,2-diacylglycerol (DAG) site of PKC or indirectly activates PKC.
 42. The kit of claim 39, wherein the PKC activator is chosen from macrocyclic lactones, diacylglycerol derivatives other than phorbol esters, isoprenoids, daphnane-type diterpenes, bicyclic triterpenoids, naphthalenesulfonamides, diacylglycerol kinase inhibitors, and growth factor activators.
 43. The kit of claim 42, wherein the PKC activator is a macrocyclic lactone.
 44. The kit of claim 43, wherein the macrocyclic lactone is chosen from bryostatin, bryologs, and neristatin.
 45. The kit of claim 44, wherein the bryostatin is bryostatin-1.
 46. The kit of claim 39,wherein the PKC activator and the anticoagulant are formulated together.
 47. The kit of claim 39, wherein the PKC activator and the anticoagulant are formulated separately.
 48. The kit of claim 39, wherein the anticoagulant composition is formulated for intravenous administration.
 49. The kit of claim 48, wherein the anticoagulant composition and the PKC activator composition are both formulated for intravenous administration.
 50. A method of treating stroke in a subject in need thereof comprising: (a) identifying a subject having suffered a stroke; (b) administering to the subject a therapeutically-effective amount of a protein kinase C (PKC) activator; (c) determining whether the subject suffered an ischemic stroke or hemorrhagic stroke; and (d) if the subject suffered an ischemic stroke, administering a therapeutically-effective amount of an anticoagulant.
 51. The method of claim 50, wherein step (c) comprises taking a computed tomography (CT) scan. 